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Characterization of recombinant fragment of an antibody against CD3 marker.
Písačková, Jana ; Maloy Řezáčová, Pavlína (advisor) ; Obšil, Tomáš (referee)
Monoclonal antibody MEM-57 recognizes CD3 antigen expressed on peripheral blood T-lymphocytes. CD3 surface glycoprotein complex associates with T-cell receptor and is responsible for the transduction of activation signal. Antibody MEM-57 has, therefore, a large diagnostic and therapeutic potential. It could be used in autoimmune diseases diagnostics, for classification of T-cell leukemias and, as an immunosuppressant, in transplantation. The most promising therapeutic use of MEM-57 antibody would be the construction of a "Bispecific T-cell Engager" (BiTE) antibody format with potential application in cancer therapy. In this format, single-chain variable fragment (scFv) of MEM-57 would be fused with an anti-tumor antigen scFv. The thesis is focused on biochemical and biophysical characterization of MEM-57 antibody scFv fragment. Recombinant antibody fragment scFv MEM-57, equipped with the pelB leader sequence, c-myc tag and His5 tag, was produced from a pET22b(+) vector into the periplasmic space of E. coli BL21 (DE3). Two-step purification protocol, employing nickel chelation affinity chromatography and ion-exchange chromatography, was developed to obtain high yield of pure protein. The antigen binding activity of scFv MEM-57 was confirmed by flow cytometry. Structural information on scFv MEM-57...
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Understanding the interaction of antibodies and transcription factors with their ligands through structural biology
Škerlová, Jana
Understanding protein function highly benefits from the knowledge of its three-dimensional structure, especially in the case of protein-ligand complexes. Structural biology methods such as X-ray crystallography, SAXS and NMR are therefore widely used for structural studies of protein-ligand interaction. In this work, these methods were used to understand two biological processes involving protein interactions: X-ray structural analysis was used to study binding of effector molecule to a prokaryotic transcription factor. NMR and SAXS techniques were used to study interaction of a monoclonal antibody with its protein antigen. Transcriptional regulator DeoR negatively regulates the expression of catabolic genes for the utilization of deoxyribonucleosides and deoxyribose in Bacillus subtilis. DeoR comprises an N-terminal DNA-binding domain and a C-terminal effector-binding domain (C-DeoR), and its function is regulated by binding of a small-molecular effector deoxyribose-5-phosphate. We determined crystal structures of C-DeoR both in the free form and in complex with deoxyribose-5-phosphate. Structural analysis revealed unique covalent binding of effector molecule through a reversible Schiff-base double bond with an effector-binding-site lysine residue. The physiological nature of this binding mode was...
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Protein engineering as a tool for the production of antibody derivatives
Šulc, Josef ; Bařinka, Cyril (advisor) ; Mikulecký, Pavel (referee)
This thesis deals with production and properties of disulfide-stabilized single-chain variable fragments of the 5D3 antibody (dsscFv), which specifically recognizes and binds to glutamate carboxypeptidase II (GCPII), an antigen closely related to the prostate carcinoma processes and other tumor diseases. Small antibody fragments are in current focus of development of diagnostic and therapeutic reagents. However, compromised stability of antibody derivatives often results in low production yield or loss of function. Introduction of structural changes by protein engineering is often used to solve the issue. The aim of the study was based on enhancement of protein stability by the introduction of interdomain disulfide bond into the structure of single-chain variable fragment. The effect of modification was evaluated by estimation of production yield and affinity of studied protein. The aforementioned antibody derivative was produced using an Escherichia coli expression system, using specific signal sequences leading the production to the bacterial periplasm. The attempted stabilization was carried out by introducing mutations at LV-G44 and HV-G100 positions, replacing glycines with cysteines. The binding affinity of the derivative for human GCPII was determined using ELISA. This thesis also shows a solved 3D...
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Understanding the interaction of antibodies and transcription factors with their ligands through structural biology
Škerlová, Jana
Understanding protein function highly benefits from the knowledge of its three-dimensional structure, especially in the case of protein-ligand complexes. Structural biology methods such as X-ray crystallography, SAXS and NMR are therefore widely used for structural studies of protein-ligand interaction. In this work, these methods were used to understand two biological processes involving protein interactions: X-ray structural analysis was used to study binding of effector molecule to a prokaryotic transcription factor. NMR and SAXS techniques were used to study interaction of a monoclonal antibody with its protein antigen. Transcriptional regulator DeoR negatively regulates the expression of catabolic genes for the utilization of deoxyribonucleosides and deoxyribose in Bacillus subtilis. DeoR comprises an N-terminal DNA-binding domain and a C-terminal effector-binding domain (C-DeoR), and its function is regulated by binding of a small-molecular effector deoxyribose-5-phosphate. We determined crystal structures of C-DeoR both in the free form and in complex with deoxyribose-5-phosphate. Structural analysis revealed unique covalent binding of effector molecule through a reversible Schiff-base double bond with an effector-binding-site lysine residue. The physiological nature of this binding mode was...
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Understanding the interaction of antibodies and transcription factors with their ligands through structural biology
Škerlová, Jana ; Maloy Řezáčová, Pavlína (advisor) ; Hrabal, Richard (referee) ; Obšil, Tomáš (referee)
Understanding protein function highly benefits from the knowledge of its three-dimensional structure, especially in the case of protein-ligand complexes. Structural biology methods such as X-ray crystallography, SAXS and NMR are therefore widely used for structural studies of protein-ligand interaction. In this work, these methods were used to understand two biological processes involving protein interactions: X-ray structural analysis was used to study binding of effector molecule to a prokaryotic transcription factor. NMR and SAXS techniques were used to study interaction of a monoclonal antibody with its protein antigen. Transcriptional regulator DeoR negatively regulates the expression of catabolic genes for the utilization of deoxyribonucleosides and deoxyribose in Bacillus subtilis. DeoR comprises an N-terminal DNA-binding domain and a C-terminal effector-binding domain (C-DeoR), and its function is regulated by binding of a small-molecular effector deoxyribose-5-phosphate. We determined crystal structures of C-DeoR both in the free form and in complex with deoxyribose-5-phosphate. Structural analysis revealed unique covalent binding of effector molecule through a reversible Schiff-base double bond with an effector-binding-site lysine residue. The physiological nature of this binding mode was...
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Characterization of recombinant fragment of an antibody against CD3 marker.
Písačková, Jana ; Maloy Řezáčová, Pavlína (advisor) ; Obšil, Tomáš (referee)
Monoclonal antibody MEM-57 recognizes CD3 antigen expressed on peripheral blood T-lymphocytes. CD3 surface glycoprotein complex associates with T-cell receptor and is responsible for the transduction of activation signal. Antibody MEM-57 has, therefore, a large diagnostic and therapeutic potential. It could be used in autoimmune diseases diagnostics, for classification of T-cell leukemias and, as an immunosuppressant, in transplantation. The most promising therapeutic use of MEM-57 antibody would be the construction of a "Bispecific T-cell Engager" (BiTE) antibody format with potential application in cancer therapy. In this format, single-chain variable fragment (scFv) of MEM-57 would be fused with an anti-tumor antigen scFv. The thesis is focused on biochemical and biophysical characterization of MEM-57 antibody scFv fragment. Recombinant antibody fragment scFv MEM-57, equipped with the pelB leader sequence, c-myc tag and His5 tag, was produced from a pET22b(+) vector into the periplasmic space of E. coli BL21 (DE3). Two-step purification protocol, employing nickel chelation affinity chromatography and ion-exchange chromatography, was developed to obtain high yield of pure protein. The antigen binding activity of scFv MEM-57 was confirmed by flow cytometry. Structural information on scFv MEM-57...
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Advances in the production of antibodies in plants
Angelis, Karel ; Šmídková, Markéta
Plants are the oldest biotechnological production system and as eukaryots have advantage over bacteria to modify expressed proteins. Laboratory model plants like tobacco along with crop plants with established logistics for harvest and storage like potatoes or rice are used for production of desired proteins. Current trend is to replace soil grown plants with liquid cultures of plant cells grown under controlled conditions. In this respect the moss Physcomitrella patens besides ability to grow in liquid culture enables due to efficient homologous recombination to modify posttranslational glycosylation to correspond human. For heterologous expression in plants, recombinant, single-chain molecules composed of only variable VL and VH regions of the original antibody forming binding pocket are constructed and expressed. Recombinant scFv antibodies are then used as a part of therapeutic or diagnostic drugs in oncology.
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Molecular cloning, E.coli expression and purification of SCFV antibody fragments of diagnostic/therapeutic interest
Král, Vlastimil ; Fábry, Milan ; Hořejší, Magdalena ; Závada, Jan ; Sedláček, Juraj
We describe molecular cloning, expression, purification and properties of two single chain antibody variable fragments (scFv) of potential diagnostic use, namely scFv M75 and scFv Tu-20. The former scFv is derived from a monoclonal antibody M75 specific for a cell surface protein MN/CA IX, strongly associated with many types of human carcinomas. The latter scFv is derived from a monoclonal antibody TU-20 specific for neuronal beta-III-tubulin.
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